The murine monoclonal antibody (MAb) A33, is an IgG2a/k which recognises a heat-stable, protease-resistant neuraminidase-resistant epitope which is homogeneously expressed in virtually all primary and metastatic colon cancers. The expression of the antigen is restricted to normal colonic mucosal epithelium and colonic carcinoma and it is not shed into the circulation (Weltet al., 1990). The antigen is expressed on a number of human tumour cell lines, including ASPC-1 and Colo205.
The A33 antibody internalises after binding to antigen. It has been shown to localise to tumours in vivo in patients with hepatic metastases of colorectal carcinoma (Welt et al., 1990). .sup.131 l labelled antibody (0.2 mg., n=3; 2mg., n=8; 10 mg. n=3; 25 mg, n=3; 50 mg, n=3; labelled with 2-5 mCi) was administered i.v. to 20 patients 7-8 days prior to surgery. Selective mAb33 localisation to tumour tissue was demonstrated in 19 of the 20 patients. One week after administration tumour/liver ratios ranged from 6.9 to 100 while tumour/serum ratios ranged from 4.1 to 25.2. Studies with a control 1251 MAb Ta99 (2 mg. dose, co-administered to three patients who received 2 mg. A33), showed that the tumour uptake of A33 was specific, with 2.3 to 45 fold higher uptake for A33.
However a human anti-mouse antibody (HAMA) response was detected in patients as early as day 7 in 8 patients and all patients developed a HAMA response by day 30. Initially the predominant HAMA was IgM but by day 30 IgG responses were also detected. The HAMA reactivity was not, however, restricted to the IgG2a isotype. Murine IgG1 and IgG3 could also be detected with patient serum. It can therefore be expected that the usefulness of the murine MAb A33 as a therapeutic agent in humans will be limited by the fact that the human subject will mount an immunological response to the MAb and will either remove it entirely or at least reduce its effectiveness.
Proposals have been made for making non-human MAbs less antigenic in humans. Such techniques can be generically termed "humanisation" techniques. These techniques generally involve the use of recombinant DNA technology to manipulate DNA sequences encoding the polypeptide chains of the antibody molecule. A simple form of humanisation involves the replacement of the constant regions of the murine antibody with those from a human antibody (Morrison et al., 1984, Whittle et al., 1987). A number of these chimeric mouse variable human constant region antibodies have been administered to patients. (Baker et al., 1991; Begent et al., 1990; Ghrayeb et al., 1991; Khazaeli A., 1992; Knox et al., 1990; LoBuglio et al., 1989; Meredith et al., 1991, 1992; Saleh et al., 1992; Trang et al., 1990). In general an immune response still develops against these chimeric antibodies although the level of the response is usually lower than that seen against the murine antibody and delayed in onset. In one case, chimeric 17-1A(.gamma.1), the response seen has been remarkably low (LoBughio et al., 1989; Meredith et al., 1991; Trang et al. 1990) with only 1 of 16 patients treated showing a low level response. In the other examples approximately 50% of the patients treated develop a response which is predominantly against the murine variable region, including the binding site. The lowering of the level of the HAMA response to the chimeric antibodies leads to the expectation that further humanisation of the variable region outside of the antigen binding site may abolish the response to these regions and further reduce the response against the binding site.
A more complex form of humanisation of an antibody involves the re-design of the variable region domain so that the amino acids constituting the murine antibody binding site are integrated into the framework of a human antibody variable region (Jones eta., 1986). That this can be cone is a consequence of the close structural and sequence relationship between immunoglobulins from different species.
Within the variable region sequence it has been noticed that a number of non-contiguous sequences, three per domain, are particularly variable (termed hypervariable). This observed sequence variation between antibodies was postulated to provide the variability which enables antibodies ,o recognise and bind to a wide range of antigenic forms and the three hypervariable regions in each domain were termed the Complementarity Determining Regions (CDR's) (Wu and Kabat, 1970; Kabat et al., 1987). This proposal has been confirmed from structural studies where it is seen that for the most part the hypervariable sequences are associated on the surface as a set of loops which form a large surface patch and that these sequences are in contact with antigen in those cases where antigen-antibody complexes have been studied (Amit et al., 1986; Bhat et al., 1990; Boulot et al., 1987, 1990; Colman et al., 1987; Davies et al, 1989; Padlan et al., 1989; Poljak, 1991; Sheriff et al., 1987). In most but not all cases the CDRs correspond to, but extend a short way beyond, these structural loop regions.
Substitution of these hypervariable regions alone into a human antibody does not, in general, lead to the reconstitution of the binding affinity of the murine antibody (Verhoeyen et al., 1988; Riechmann et al., 1988).
Residues not identified in the loop or hypervariable regions must therefore contribute to antigen binding directly or indirectly by affecting antigen binding site topology, by inducing a stable packing of the individual variable domains, or by stabilising the inter-variable domain interaction. Methods for the identification of these key framework locations are available (eg. Adair et al., 1991; Kurrie et al., 1990; Law et al., 1991; Padlan, 1991; Queen et al., 1990; Winter, 1987).
The choice of human framework for the humanisation process can be based on the desire to use an antibody domain for which there is a known structure determined from X-ray crystallography, so that some positional information is available about framework amino acids, or to use a matched light and heavy chain pair, or to use a representative example from the various human subgroups, or simply to search the available human sequences and identify human antibody domains which have high homology to the variable domains of the mouse antibody in question.
Humanisation has led to the reconstitution of full antigen binding activity in a number of cases (Co et al., 1990, 1992; Carter et al., 1992; Routledge et al., 1991; and International Patent Specifications Nos. WO 91/09967; WO 91/09968; and WO 92111383).
The reduction in immunogenicity that may be expected from the humanisation process has been examined by Hakimi et al., (1991) using the humanised form of the anti-Tac antibody, anti-Tac-H (Queen et al., 1989). The humanised antibody was expressed in the murine myeloma line Sp2/0. The antibody contained less than 10 endotoxin units/mg of protein. Antibody was administered to 8 groups each of 4 cynomolgus monkeys. The groups were given either anti-Tac-H or the murine antibody (anti-Tac-M) i.v. in doses of 0, 0.05, 0.5 or 5 mg/kg each day for 14 days followed by challenge with the same antibody on day 42. Pharmacokinetics and immunogenicity were monitored throughout the study. Adverse responses (anaphylaxis) to the anti-Tac-H on the 42 d re-challenge was seen in one animal given 5 mg/kg and in all 4 animals given 0.05 mg/kg of anti-Tac-M. Therefore none of the animals given 0.5 or 5 mgikg of anti-Tac-M received the day 42 re-challenge.
The response to the humanized antibody was seen to be lower in absolute amount and delayed in onset compared to the response to the murine antibody. In the anti-Tac-M groups response in 9 of 12 animals was seen during the course of treatment, while in those animals given the humanized antibody response was generally seen 5 to 10 days after the last injection. For both antibodies the level of response appeared in general to be inversely proportional to the dosage. The earliest and most vigorous response to the murine antibody was seen in the 0.05 mg/kg group where in the case of one animal an anti-antibody response measured at over 200 mg/mL was seen. In contrast the groups receiving the higher doses of the murine antibody had similar, lower patterns of absolute levels of anti-anti-Tac-M, with the 5 mg/kg group showing a delayed onset of response. In two animals no response was seen at this dose, while in the 0.5 mg/kg group one animal showed no response. For the animals given the humanized antibody a similar trend was seen but the levels of response were much lower. The highest response was seen in one animal given 0.5 mg/kg. where 60 mg/mL of anti-antibody was seen at day 42. This level of response was exceeded by 9 of the 12 animals given the murine antibody. Again the response was inversely proportional to the dosage with all of the animals in the 0.05 mg/kg group showing some level of response (in the range 5 to 25 mg/mL of anti-antibody). In all cases where a second dose at 42 days was able to be given a large, &gt;10 fold, increase in specific titre was observed.
The type of response was measured by competition ELISA in which each of the murine or humanized antibodies was bound to a solid phase and incubated with serum in the presence of various potential competitors, including the antibodies themselves, soluble IL-2R or non-specific murine or human IgG. It was shown that the response to both murine and humanized antibody was both anti-idiotypic and anti-isotypic. However the anti-isotype response against the humanized antibody was marginal. The majority of the response against the humanized antibody could be inhibited by the presence of humanized or murine anti-Tac or soluble IL-2R suggesting that the response was predominantly anti-binding site.
Three studies of the use of the humanized anti-CAMPATH-1 antibody, CAMPATH-1H, have been described (Crowe, 1992: Hale et al., 1989; Mathieson et al., 1990). In the first study two patients with non-Hodgkin lymphoma were treated with CAMPATH-1H for up to 43 days with escalating doses ranging from 1-20 mg/day by intravenous infusion (Hale et al., 1988). No anti-antibody response was detected during the course of treatment. The lack of response may be due to the fact that the antibody itself is immunosuppressive and the patients were already immunosuppressed as a result of their disease (Hale et al., 1988).
A further case study has been reported (Mathieson et al., 1990). The CAMPATH-1 H antibody has been used in conjunction with a rat anti-human CD4 to establish a remission in a patient with a chronic and previously intractable systemic vasculitis. The lymphocyte population was depleted by a 3 day treatment of CAMPATH-1 H (i.v. 2 mg/day) followed by the rat anti-human CD4 for 12 days at 20mg/day (i.v.) to remove any remaining T.sub.H cells. After the treatment course remission of disease occurred that has lasted 12 months. No anti-antibody response has been detected.
More recently it has been disclosed that a further 8 patients with rheumatoid arthritis have been treated with CAMPATH-1 H. The patients were given 4mg/day for 5 days followed by 8 mg/day for a further 5 days. In 7 of 8 cases there was statistically significant reduction in assessable criteria for pain and joint disease (Crowe, 1992). No anti-antibody response was evident for a period of several months post treatment.
Hird et al., (1991) have administered the humanised anti-PLAP antibody Hu2PLAP (Verhoeyen et al., 1991), derived from the murine antibody H17E2 (Travers and Bodmer, 1984) in 7 patients with various carcinomas, including ovarian, stomach and breast. Two patients had raised serum levels of human anti-mouse antibody prior to therapy. In one case the patient had previously been treated with the murine antibody HMFG1, .sup.90 Y labelled by conjugation with the macrocycle DTPA. The second patient had had prior treatment with murine H17E2, .sup.111 In labelled by conjugation with the macrocycle, DOTA (Moi et al., 1988). The 7 patients received 220-833 mg of Hu2PLAP-.sup.111 In-DOTA i.v., as a radio-imaging dose. The patients were monitored at 24 and 96 h. In those patients with no pre-existing HAMA response the t.sub.1/2 b was 73.1 h compared to 27 h for patients given the murine antibody. Where pre-existing HAMA response was seen the patients showed t.sub.1/2 b of 47 h and 39 h. None of the 7 patients, however developed antibodies specific for the Hu2PLAP over the 96h of study, although 3 patients developed anti-DOTA antibodies, one of these being the patient previously treated with murine H17E2-.sup.111 In-DOTA and who had a pre-existing anti-DOTA response.